ABSTRACT Cystic Fibrosis (CF), the most common recessive disease among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a cAMP-activated chloride ion channel. Ninety percent of CF patients carry at least one copy of the ?F508 allele. Recent studies have shown that two small molecules, the folding corrector VX-809 and the potentiator VX-770, act in combination and partially restore ?F508-CFTR ion channel function in human bronchial epithelial (HBE) cells. However, these compounds are only marginally effective for patients with the ?F508 mutation. A key limitation is that most CF patients produce high levels of Transforming Growth Factor (TGF)-?1. Our published work showed that clinically relevant levels of TGF-?1 repress ?F508-CFTR transcription in HBE cells, acting upstream of modulators to block rescue of ?F508-CFTR. High TGF-?1 levels also prime CF patients for inflammation, epithelial-mesenchymal transformation (EMT), and fibrosis. TGF-?1 initiates signal transduction by stimulating the constitutively active TGF-?1 receptor (T?R)-II to interact with and phosphorylate T?R-I at the plasma membrane. By contrast, Protein Phosphatase 1 (PP1) protects T?R-I from constitutive activation by T?R-II in non-stimulated cells. It is unknown how TGF-?1 blocks PP1 interaction with T?R-I to activate signaling. Our preliminary work in HBE cells indicates that the scaffold organized by Lemur Tyrosine Kinase (LMTK2) at the basolateral plasma membrane favors TGF-?1 signaling by inactivating the catalytic subunit of PP1 (PP1C), thus allowing activation of T?R-I and signal transduction. Moreover, our data indicate that activating PP1C blocks TGF-?1 signaling in HBE cells. Our central hypothesis is that TGF-?1 stabilizes the LMTK2 scaffold to activate signaling leading to inflammation, fibrosis, and transcriptional repression of ?F508- CFTR in HBE cells. In so doing, LMTK2 allows TGF-?1 to antagonize ?F508-CFTR protein rescue by small molecules, and worsens outcomes. Targeting the LMTK2 scaffold thus represents a novel therapeutic strategy for CF to control TGF-?1 signaling, attenuate inflammation and fibrosis, and facilitate rescue of ?F508-CFTR in HBE cells. In Aim 1 we will examine TGF-?1 effects on the protein-protein interactions between T?R-I, LMTK2, and PP1C in HBE cells. In Aim 2, we will test whether TGF-?1 recruits and/or activates LMTK2 at the basolateral plasma membrane in HBE cells. In Aim 3, we will elucidate whether TGF-?1 signaling can be attenuated by blocking LMTK2 inactivation of PP1C in HBE cells. We will use state-of-the-art research tools. Because TGF-?1 signaling is cell-type and cell-context dependent we will use HBE cells expressing ?F508- CFTR, which exhibit many of the characteristics associated with CF airway disease in vivo and are an ideal model for pre-clinical experimentation. We anticipate that our studies will lead to novel therapy targeting excessive TGF-?1 signaling triggered by high TGF-?1 levels present in most CF patients, to preserve airway integrity, and to allow small molecules to restore the ?F508-CFTR function.